Cell-Impermeable Buffering Fluorogenic Probes for Live-Cell Super-Resolution Imaging of Plasma Membrane Morphology...

Area of Science:

• Cell Biology
• Biophysics
• Microscopy

Background:

• Super-resolution fluorescence imaging offers nanoscale insights into plasma membrane (PM) structure and function.
• Limitations in probe photostability and cell internalization hinder super-resolution imaging of dynamic PM processes.

Purpose of the Study:

• To develop novel fluorogenic probes for enhanced super-resolution imaging of the plasma membrane.
• To investigate plasma membrane dynamics, including extracellular vesicle formation and fusion.

Main Methods:

• Assembly-mediated buffering fluorogenic probes (BMP-14 and BMP-16) were synthesized and characterized.
• Structured illumination microscopy (SIM) was employed to visualize PM dynamics.
• Single-molecule fluorescence imaging techniques (e.g., PAINT) were utilized.

Main Results:

• BMP-14 and BMP-16 demonstrated rapid PM labeling and prolonged retention (>2 h).
• These probes enabled stable, long-term super-resolution imaging by rapidly replacing photobleached fluorophores.
• Dynamic processes of PM filopodia movement, extracellular vesicle (EV) formation, and distinct EV fusion modes were observed.
• BMP-16 showed single-molecule fluorescence blinking, suitable for PAINT imaging.

Conclusions:

• The developed PM-buffering probes overcome limitations in photostability and labeling for super-resolution microscopy.
• Novel insights into EV formation and fusion mechanisms at the plasma membrane were obtained.
• BMP-16 offers a unique capability for advanced cell membrane imaging techniques like PAINT.